The Research About Protective Effect Of Cynomorium Songaricum Rurp Polysaccharide On H₂O₂-induced Oxidative Damage In PC12 Cell

Cynomorium songaricum Rurp polysaccharide?CSP?is a medicinal plant polysaccharides extracted from Gansu characteristic plant Cynomorium songaricum Rurp.It's biological activity is very broad,antioxidant activity in vitro as one of an important biological activity has been confirmed.The oxidative damage model on cell was established in this paper,The protective effect and mechanisms of Cynomorium songaricum Rurp polysaccharide on H₂O₂-induced oxidative damage in PC12 cell was researched,it lay a theoretical foundation for the study of the natural drug of the antioxidation.In this study,Cytotoxicity of H₂O₂ and CSP on PC12 cells was measured by Alarma Blue assay,the optimum damage concentration and protection concentration was selected;The changes in the nucleus was watched by Hoechst33258 staining;The cell cycle was detected by propidium iodide staining with flow cytometry assay;apoptotic cells was detected by Annexin V-FITC / PI double staining and flow cytometry;The content of 8-hydroxy-deoxyguanosine?8-OHd G?was measured by double-antibody sandwich method;The level of LDH release was detected by spectrophotometry;the changes of ROS content in cells was detected by the fluorescence spectrophotometry;The mitochondrial membrane potential was detected by using fluorescent probe JC-1 though fluorescence microscopy and fluorescence spectrophotometer;The concentration of calcium ion in cells was measured by using fluorescent probe Fura-2 AM though multifunctional microplate reader;The activity of Caspase-3,Caspase-9,SOD,GSH-Px enzymes in cells and MDA levels outside the cell was detected by spectrophotometry.The results show that:1.H₂O₂ can induce PC12 cells oxidative dose-dependently and time-dependently.The final concentration of H₂O₂ is 500?mol / L on PC12 cells for 24 h,cell could be injuried and the viability be decreased,activity of ROS be increased,levels of LDH release be increased,mitochondrial membrane potential be decreased significantly,calcium concentration of PC12 cells be increased,caspase pathway was activated,activity of caspase-3 caspase-9 be increased,thereby further apoptosis of PC12 cells be induced.In addition,ROS can also cause DNA damage,leading to cell cycle arrest.H₂O₂ can induce PC12 cells oxidative damage and apoptosis,it may be associated with increased ROS,intracellular calcium concentration,mitochondrial and caspase pathway.2.After pre-incubation Cynomorium songaricum Rurp polysaccharide?CSP?for 24 h,then added to a final concentration of H₂O₂ 500?mol / L,the PC12 cell damage caused by H₂O₂ can be significantly reduced,cell viability be increased,the ROS content also be decreased.LDH release was decreased.mitochondrial membrane potential be incteased significantly.The concentration of intracellular calcium was decreased significantly.Caspase pathway was inhibited.The apoptosis of cell was inhibited.Cell cycle resistance stagnation was prevented.It directed that 0.25-1mg / ml of CSP has a protective effect on PC12 cells against H₂O₂-induced oxidative damage,the mechanism may be related to the antioxidant,repairing of mitochondrial damage,change of cell cycle constitute and inhibition of activation of mitochondrial and caspase apoptosis pathway.In summary,The studies of experiment have shown that: Cynomorium songaricum Rurp polysaccharide has significant protective effect on PC12 cell indury induced by H₂O₂ in a certain dose range,its protective effect may be related to clear ROS,mitigate oxidative DNA damage and inhibition of apoptosis mediated by mitochondrial pathway.